4C-4D Flashcards
What is Polymerase Chain Reaction (PCR)
Artificial form of DNA replication
Describe Taq Polymerase
Enzymes found in bacteria that can withstand high heat temperatures and can be used to synthesise DNA or RNA
IDEAL TEMP = 72C
Describe DNA primers
bind to single stranded DNA providing a starting point for DNA synthesis.
What is buffer solution?
Suitable chemical environment for taq polymerase by maintaining the correct pH
Define primers
Single stranded short chains of nucleotides that are complementary to the template strand. Allows taq polymerase to extend the DNA strand
Define forward primers
Binds to start codon 3’ end of template strand letting taq polymerase synthesise DNA strand in RNA polymerase would function
Define Reverse primers
Bind to stop codon at 3’end. Taq polymerase syntheisses new DNA strand in reverse direction that RNA polymerase would function
What are 3 steps of PCR
- DENATURATION
- ANNEALING OF PRIMERS
E3. XTENSION
Describe Denaturation and its optimal tempreture
90-95C. Hydrogen bonds between 2 strands of DNA are broken
Describe Annealing of primers and its optimal tempreture
50-55C. Temp lowered allowing primers to bind (anneal) to complementary bases on each single strand of DNA
Describe Extension and its optimal tempreture
Taq polymerase extends the DNA strand from the primers
What is Gel Electrophoresis?
Separating segments of DNA according their lengths
Describe the steps of Gel Electrophoresis?
- DNA samples placed into wells at one end of a slim slab of agarose gel
- Electrical current passed through the gel
- *DNA molecule contains a negatively-charged phosphate group, so DNA is attracted to the positive electrode. Hence wells are closest to the negative electrode - The molecules diffuse through the gel, and smaller lengths of DNA move faster than larger lengths.
- Lights turned off, flueorscent dye used to dye the DNA fragments so the DNA is visible
Smallest molecules go to the bottom
Describe DNA profiling
The process where a specific DNA pattern, called a profile, is obtained from a person or sample of bodily tissue
Describe the steps of DNA profiling
- A DNA sample is collected (e.g. from blood, semen, saliva, etc.) and then amplified using PCR.
- DNA (with STR sequences) are cut with specific restriction enzymes to generate fragments.
- Fragment length will differ between individuals due to the variable length of their short tandem repeats.
- The fragments are separated using gel electrophoresis and the resulting profiles are compared.
What are STR’s
Consist of a variable number of tandem repeats of a 2 to 6 base pair sequence
